The present invention relates to liposome formulations that are physically stable. In particular the present invention relates to steric stabilization of cationic liposomes by an unique film method whereby glycolipids are incorporated into the liposomes. The stabilized liposomes can be used either as an adjuvant for antigenic components or as a drug delivery system. In particular the invention relates to vaccines with adjuvants in aqueous media for immunization, where the final product is stable.
The first vaccines used in humans to produce immunity against infectious diseases consisted of live, attenuated pathogens. The attenuated forms were either naturally occurring closely related organisms or obtained through serial passages in culture. One example is tuberculosis that is combated by vaccination with attenuated but living strains of Mycobacterium bovis (BCG vaccine). However, the efficacy of this procedure does not always provide satisfactory resistance to human tuberculosis in every population. There is therefore a need for new and efficient ways of producing immunity against tuberculosis and other infectious diseases. A particular promising approach has been to isolate and use recombinant forms of immunodominant antigens such as the early secretory antigenic target (ESAT-6) and antigen 85 (Ag85) as a vaccine. These vaccines are well-defined and side-reactions are minimized. Unfortunately, many highly purified substances, e.g., purified recombinant proteins, are not very immunogenic and do not produce an effective immune response protective against the real infectious disease. This fact is well known and many attempts have been made to increase the immunogenic properties by combining the substance in question with so-called adjuvants. Depending on the pathogen, protection may require that either a humoral or a cell-mediated response predominate. The development of a specific kind of immune response (humoral or cell-mediated) can be determined by the choice of adjuvant.
Protective immunity against an intracellular pathogen like M. tuberculosis requires a cell-mediated immune response, and a suitable adjuvant for a subunit vaccine directed against TB should enhance a Th1 response (Lindblad et. al., 1997). It is generally believed that antibodies do not play an important role in immunity to TB whereas cell-mediated release of IFN-gamma (interferon gamma) is the most important cytokine involved in protection (Collins & Kaufmann, 2001).
A large number of adjuvants that induce a cell mediated immune response have been suggested but in general without any being ideal in all respects.
One particular effective type of adjuvant that promotes a cell-mediated immune response is quaternary ammonium compounds, such as dimethyldioctadecylammonium (DDA) (Hilgers and Snippe, 1992). DDA is a synthetic amphiphile comprising a hydrophilic positively charged dimethylammonium head-group and two long hydrophobic alkyl chains. In an aqueous environment DDA self-assemble to form vesicular bilayers similar to liposomes made from natural phospholipids. Combinations of DDA and other immunomodulating agents have been described. Administration of Arquad 2HT, which comprises DDA, in humans was promising and did not induce apparent side effects (Stanfield et. al., 1973). An experimental vaccine based on culture filtrate proteins from M. tuberculosis and DDA generated a protective immune response against TB in mice (Andersen, 1994). Vaccination of mice with a fusion protein of M. tuberculosis proteins ESAT-6 and Ag85B, and DDA/MPL as adjuvant, provides protection similar to that obtained by BCG vaccination (Olsen et. al., 2001). These studies demonstrate that, in contrast to e.g., alum, DDA-based adjuvants are able to induce a protective immune response against TB in mice. Moreover, DDA has been used as an adjuvant for a DNA vaccine against pseudorabies virus leading to enhanced T-cell responses is and anti-viral immunity (van Rooij et. al., 2002).
Addition of TDM (alpha,alpha′-trehalose 6,6′-dimycolate) oil emulsions to DDA solutions was investigated by Woodard et. al. (1980) as adjuvants for Brucella abortus vaccines based on heat killed bacteria. Neither DDA alone nor the mixtures of DDA and TDM was able to induce protection. In another study of a Brucella abortus subunit vaccine based on a soluble protein extract, a combination of DDA and TDM was also used as adjuvant (Dzata et. al., 1991), and the mixture was found to enhance the immune responses (antibody levels, skin test response, and IL-2 levels) observed compared to DDA alone. Holten-Andersen et. al. (2004) studied a combination of DDA liposomes and a suspension of TDB (alpha,alpha′-trehalose 6,6′-dibehenate), and administration of the ESAT-6 antigen with this adjuvant mixture was found to induce a strong protective immune response against tuberculosis which was significantly higher than when ESAT-6 was administered in DDA liposomes.
Unfortunately, suspensions of amphiphilic quaternary ammonium compounds such as DDA alone or mixtures of DDA and MPL, TDM or TDB as described above are physically unstable and prolonged storage at 4° C. is not possible without the occurrence of aggregation and precipitates. As precipitation will prevent clinical use of the formulation, the lack of stability of DDA formulations has so far been a major obstacle for any application in humans.
In Great Britain Pat. No. 2147263-A, Takahashi and Tsujii describe stabilization of vesicles from quaternary ammonium compounds by mixing two quaternary ammonium compounds together or adding various detergents to the quaternary ammonium compound.
In U.S. Pat. No. 5,026,546, Hilgers and Weststrate describe stabilization of an adjuvant suspension of DDA with a polymer of acrylic acid crosslinked with polyallyl sucrose.
Lyophilization of cationic lipid-protamin-DNA complexes for transfection of cells was described by Li et. al. (2000). The effect of adding traditional cryoprotectants like monosaccharides and disaccharides was evaluated, and disaccharides were found to preserve particle size better than monosaccharides. Also non-lyophilized lipid-protamin-DNA complexes stabilised with 10% sucrose maintained a stable particle size after 8 weeks storage at 4° C., but the transfection efficiency was higher in lyophilized than in non-lyophilized samples.
U.S. Pat. No. 5,922,350 describes a method for extending storage of liposomes e.g., based on phospholipids by adding sugars like trehalose and sucrose before the dehydration of the liposomes. Furthermore, the patent describes that delayed loading of the preformed, stored liposomes is feasible by a combination of concentration gradients and the dehydration-rehydration process.
Liposomes of phospholipids for drug delivery (fusogenic liposomes) stabilized with a polyethylene glycol derivative are described in WO 96/10392. Another drug delivery formulation described in WO 02/03959 discloses a formulation comprising cationic liposomes and neutral liposomes where each liposome group either carries the same or different therapeutic agents.
Preferred methods for making liposome preparations are described by Bangham (Bangham et. al., 1965). This preparation involves dissolving phospholipids in an organic solvent which is then evaporated to dryness leaving a thin lipid film on the inside of the test tube. The dry lipid film is then hydrated in an appropriate amount of aqueous phase and the mixture is heated to above the phase transition temperature of the lipids and allowed to “swell”. The resulting liposomes which consist of multilamellar vesicles (MLV's) are dispersed by shaking the test tube. The lipids constituting the vesicular bilayer membranes are organized such that the hydrophobic hydrocarbon “tails” are oriented toward the center of the bilayer while the hydrophilic “heads” orient towards the in- and outside aqueous phase, respectively. This preparation provides the basis for producing unilamellar vesicles (UV) by methods such as sonication (Papahadjopoulos et. al., 1967) or extrusion as described by Cullis et. al. in U.S. Pat. No. 5,008,050.
Other techniques used to prepare vesicles are reverse-phase evaporation introduced by Szoka and Papahadjopoulos (Szoka and Papahadjopoulos, 1978; U.S. Pat. No. 4,235,871). This technique consists of forming a water-in-oil emulsion of lipids in an organic solvent and an aqueous buffer solution containing a substance to be encapsulated. Removal of the organic solvent under reduced pressure produces a viscous gel. When this gel collapses an aqueous suspension of lipid vesicles are formed.
Another method described by Carmona-Ribeiro and Chaimovich (Carmona-Ribeiro and Chaimovich, 1983) involves injecting an organic e.g., chloroform, methanol, ethanol, solution of the desired lipids into an aqueous buffer where the lipids spontaneously forms liposomes as the solvent evaporates.
The liposomes can also be prepared by the aqueous heat method as described for DDA by Holten-Andersen et. al. (2004) by which a suspension of the liposome forming compound in aqueous buffer is heated to e.g., 80° C. by intermittent shaking for 20 minutes followed by cooling to room temperature.
Above mentioned “aqueous heat method”, used and described by Woodard et. al. (1980), Dzata et. al. (1991) and Holten-Andersen et. al. (2004) does not stabilize solutions of DDA and TDB.
In one particular preferred method protein antigens are entrapped within preformed vesicles by the dehydration-rehydration method (Kirby and Gregoriadis, 1984) in which an oligonucleotide, peptide or protein present in the aqueous phase is entrapped by freeze drying followed by rehydration of the lyophilized liposomes.
Alternatively the antigen is incorporated using the freeze and thaw technique described by Pick (Pick, 1981) and by Bally et. al. in U.S. Pat. No. 4,975,282. In this technique vesicles are mixed with the protein antigen and repeatedly snap frozen in liquid nitrogen and warmed to temperatures above the main phase transition temperature of the relevant lipids. The vesicles may be further processed to remove any non-entrapped antigen e.g., by washing and centrifuging.
It has been shown that acylated glycosides such as TDB and cord factor isolated from the mycobacterial cell wall, TDM, inhibits fusion between phospholipid vesicles (Spargo et. al., 1991 and Crowe et. al., 1994). The hydrophilic trehalose moiety is likely to be immobilized at the surface of the vesicles, thus increasing the hydration force that is an important primary barrier to fusion. Alternatively the immobilized trehalose moiety might act as a steric barrier to fusion (Spargo et. al., 1991).
Liposomes from phospholipids (without TDB) are presently used experimentally as adjuvants in e.g., influenza vaccine (Ben-Yehuda et. al., 2003). Another example is IMUXEN™ liposomal vaccine against influenza (Lipoxen Technologies Ltd.; Gregoriadis et. al., 1999).
As quaternary ammonium compounds and especially DDA is a very promising candidate for an effective vaccine adjuvant but has the major disadvantage of being physically un-stable in aqueous solution forming aggregates and precipitates during storage it is much needed to stabilise the vesicles formed. The present invention describes a new method of stabilizing adjuvant formulations composed of cationic lipids such as DDA. Additionally, by this method the adjuvant effect of the formulation is enhanced.